Molecular architecture and electron transfer pathway of the Stn family transhydrogenase

Abstract

The challenge of endergonic reduction of NADP⁺ using NADH is overcome by ferredoxin-dependent transhydrogenases that employ electron bifurcation for electron carrier adjustments in the ancient Wood-Ljungdahl pathway. Recently, an electron-bifurcating transhydrogenase with subunit compositions distinct from the well-characterized Nfn-type transhydrogenase was described: the Stn complex. Here, we present the single-particle cryo-EM structure of the Stn family transhydrogenase from the acetogenic bacterium Sporomusa ovata and functionally dissect its electron transfer pathway. Stn forms a tetramer consisting of functional heterotrimeric StnABC complexes. Our findings demonstrate that the StnAB subunits assume the structural and functional role of a bifurcating module, homologous to the HydBC core of the electron-bifurcating HydABC complex. Moreover, StnC contains a NuoG-like domain and a GltD-like NADPH binding domain that resembles the NfnB subunit of the NfnAB complex. However, in contrast to NfnB, StnC lost the ability to bifurcate electrons. Structural comparison allows us to describe how the same fold on one hand evolved bifurcation activity on its own while on the other hand combined with an associated bifurcating module, exemplifying modular evolution in anaerobic metabolism to produce activities critical for survival at the thermodynamic limit of life.

Fig. 1. Molecular architecture of StnABC.

a Three-dimensional segmented cryo-EM density of the tetrameric complex coloured by subunits. b Corresponding views of the StnABC atomic model in cartoon representation. The tetramer is mediated by the interactions of StnC subunits.

Fig. 2. Cofactor organization in StnABC complex.

a Two views of StnABC functional protomer. StnA contains one [2Fe2S]-cluster; StnB contains FMN and NAD⁺ cofactors, including three [4Fe4S]-clusters, one [2Fe2S]-cluster, and a zinc atom; StnC harbours six [4Fe4S]-clusters and one [2Fe2S]-cluster with FAD and NADPH cofactors. b The overall cofactors arrangement in StnABC tetrameric complex with distances between the neighbouring protomers marked. The distances are outside the physiological range of electron transfer between each protomer. c Overview of cofactor organisation and the edge-to-edge distances in Ångströms of StnABC complex. An electron transfer path is indicated from the FAD-NADPH site in the StnC to FMN in the StnB subunit. The reduction of NAD⁺ takes place at FMN, and Fd is reduced at the C-terminus of StnB.

Fig. 3. Mutational analysis of the StnC subunit.

a Surface representation of StnC with six [4Fe4S]-clusters and one [2Fe2S]-cluster, along with the FAD-NADPH binding site shown. The oxidation of NADPH triggers the electron flow from FAD in StnC to the FMN in StnB. b, c Zoom-ins of the FAD binding site and C7 cluster encased around cryo-EM density. d NADPH binds at the pocket carved by two Rossmann folds in the StnC subunit. e Comparison of the FAD binding site in StnC and NfnB subunits. Residue R201 in NfnB forms a hydrogen bond with N5 of FAD. The corresponding R239 in StnC also participates in hydrogen bonding but not with the N5 of FAD. f, g Transhydrogenase activity assays performed for different complex variants probing the roles of R239, K170, and C114. Fd_ox-dependent NADPH:NAD⁺ oxidoreductase activity of StnABC-His in comparison to variants StnC_R239A, StnC_R239K, StnC_K170R, StnC_K170A, StnC_K170C and StnC_C114A. Activities are the mean ± s.e.m. of three independent biological replicates, measured in triplicates (n = 3). Activities are given in U/mg. One Unit is defined as the transfer of 2 µmol electrons/min.

Fig. 4. FMN-NAD⁺ binding and mutational analysis of the electron bifurcating StnAB module.

a, b Structural model of StnAB subunit, with the corresponding cryo-EM density enveloping the FMN-NAD⁺ pair. The distances of nearby A1 and B2 clusters to the FMN are shown. c Superposition of FMN binding sites in StnB, HydB, and Nqo1. All three subunits harbour and stabilize the bound FMN using the conserved glycine-rich loop. d Comparison of the NADPH:MV_ox oxidoreductase activity and Fd_ox-dependent NADPH:NAD⁺ oxidoreductase activity of StnABC-His and variant StnABC_∆A1. Activities are the mean ± s.e.m. of three independent biological replicates, measured in triplicates (n = 3). Activities are given in U/mg. One Unit is defined as the transfer of 2 µmol electrons/min. e Overlay of the StnAB module structure determined under the StnABC_S1 and StnABC_S2 state of the enzyme. The loop (residue 182–191) undergoes a slight conformational change upon the binding of NAD⁺ in the StnABC_S2 state of the enzyme. f Comparison of the electron transfer path in the StnAB module with HydBC and Nqo1-3 subunits. In all three cases, the FMN acts as a mediator to transfer the electrons sequentially to nearby clusters. All the distances given are in Ångströms (Å).

Fig. 5. Putative mechanism of electron bifurcation in the StnABC transhydrogenase.

The reaction is initiated after NADPH binding and its oxidation at the FAD cofactor in the GltD domain of StnC. During the first round of NADPH oxidation, the electrons sequentially (one by one) travel along a chain of [4Fe4S]-clusters to reduce the FMN in StnB. The distinct iron-sulphur cluster environment surrounding the FMN cofactor in StnB is responsible for the electron bifurcating reaction of the complex. The FMN transfers the electrons to the clusters B2 and A1. The reduction of the B2 and C1 clusters triggers the binding of NAD⁺ at FMN. Another round of NADPH oxidation transfers two electrons to the binding site of the nucleotide, where FMN performs a hydride transfer to produce NADH. The reduction and dissociation of NADH triggers local rearrangements that lead to the outward “open” movement of StnA. This allows electron transfer from B2 and A1 to the clusters B3-B4 in the C-terminal domain of StnB, which is the putative binding site for Fd reduction. The StnA subunit transits to a “closed” state upon the oxidation of cluster A1, preventing the backflow of electrons from the C-ter of StnB to FMN. The resulting conformational dynamics create a redox-driven kinetic gate, which ultimately enables the reduction of Fd. The conformational changes in StnAB are thermodynamically driven by the reduction and dissociation of the nicotinamide.